Background: The main objective of this study was to isolate and identify moderately halophilic bacteria that produce saline- tolerant PULLULANASE enzyme in hypersaline condition.Methods: The bacteria were isolated by saline culture from saline soils in Degh Biarjemand desert of Shahrod. Then moderately halophilic bacteria were isolated. On the next step, the isolated halophilic bacteria were analyzed for production of pullulanse enzyme. After that, they were identified by molecular and biochemical methods.Results: A total of 11 strains were isolated from Biarjemand desert of Shahrod. 9 out of the 11 halophilic bacteria were moderately halophilic and the two others were halotolerant, but no extreme halphilic bacteria were among the isolated strains. The halophilic bacteria producing hydrolytic enzymes were analyzed. 2 strains of the bacteria produced PULLULANASE which one of them was moderately halophilic bacterium. The halophilic bacterium producing pullulanse enzyme was identified concerning the 16S rRNA sequencing technique as well as it’s molecular and biochemical properties showed that the isolate belongs to the genus Bacillus.Conclusion: In this study, for the first time, the halophilic bacteria from Degh Biarjemand desert of Shahrod were isolated and analyzed for production of PULLULANASE enzyme and special halophilic bacteria were isolated. The isolated halophilic bacterium produced saline-tolerant PULLULANASE enzyme, was tolerant and active in hypersaline condition.

Pullulanse is an enzyme that can be Produced by K.Pneumoniae, Bacillus acidopullulyticus and e few other Bacteria. This Enzme can brak pullulan biopolymer specifically and it is the most reliable method in this polymer detection. A suppernatant of K.Penamonia culture those cotains PULLULANASE used in detection and measurment of pullulan. At first, K.Penamonia was cultured in a preliminaty active nutrient agar containing pullulan (the enzyme specific substrate ) for 24 hours .Then 2ml of this culture was added to the 16ml of a culture media and it was incubated in 370 C and 120 rpm.The Enzymatic activity of this strain was determined at 5 hour intervals and the activity was seen after 10 hours the enzymatic activity of produced PULLULANASE measured according to released maltotrise in gram/lit, and 32.43g/l of maltotriose were release after 10 hours. The PH=5 and 400 C are the optimimum condition for enzymes activity.

A pullulans classified under the family Dothideales. In present study different strains isolated from nature and the best strain with a large amount of pullulan production (51) is selected for optimization. Amount of pullulan and its purity determind by PULLULANASE enzyme activity. Sucrose and glucose select as source of carbon and this strain produced 40 g/l pullulan on sucrose. The best yield was obtained on 5% sucrose. Production of pullulan under different nitrogen source (yeast extract, ammonium sulphate, sodium nitrate), pH (4- 9), temperature (25, 28, 32°C) and shacking (120, 180, 200 rpm) by this strain were investigated. Under the best condition 48g/l pullulan was produced within 5 days. A small pigment in the medium was observed during the. fermentation.

Identification and use of more efficient enzymes in the food and pharmaceutical industries is the focus of many researchers. The aim of this study was to search for a new bacterial strain capable of producing high levels of PULLULANASE applicable to biotechnology, the starch bioprocessing and food industries. A new pullulan hydrolyzing Bacillus strain was isolated and designated SDK2. Morphological and biochemical tests identified the strain as a putative Bacillus cereus strain, which was further characterized and confirmed through 16s rRNA sequencing, and was submitted to GeneBank, under the accession number FR6864500. Quantitative analysis of the strain’s PULLULANASE activity was carried out by the Dintrosalicyclic (DNS) acid-based assay. Thin layer chromatography (TLC) of the culture supernatant, identified the extracellular PULLULANASE as neoPULLULANASE. Effects of temperature and pH on PULLULANASE activity were also studied. The optimum conditions for enzyme activity, as represented by 60oC and a pH of 7, resulted in an activity of 13.43 U/ml, which is much higher than some of the previously reported activities. However, growth of B. cereus SDK2 was also observed at a pH range of 5 to 10, and temperatures of 30oC to 50oC. The effect of metal ions and reagents, such as Mg+2, Ca+2, Zn+2, Cu+2, Fe+2, Ni+2 on enzyme activity showed that Ca+2 ions increased pullulan activity, whereas the other ions and reagents inhibited PULLULANASE activity. The ability of B. cereus SDK2 to produce high levels of neoPULLULANASE stable at 60oC that can generate panose from pullulan, make this newly isolated strain a valuable source of debranching enzyme for biotechnology, the starch bioprocess and medical industries.

Aran-Bidgol salt lake is one of the lakes in central desert zone of Iran and is largest playa in Iran. This lake has shape as like a triangle that top it is towards north. Base length this triangle is 35 Km and high it is 38 Km. Screening bacteria from different points Aran-Bidgol salt lake, led to the isolation of 61 Gram-positive halophilic bacteria and 22 Gram-negative bacteria able to produce different hydrolytic enzymes. These strains are able to grow optimally in media with 5-15 % salts, 35-37°C temperature and pH 7.2. Strains No. 32, 20, 27, 40, 40, 9, 11, 24, 16 and 20 were produced amylase, protease, lipase, DNase, inulinase, xylanase, carboxy methyl cellulase, pectinase, PULLULANASE and chitinase. It is interesting that combined hydrolytic activities have been detected in some strains. DNase, indulines were the most enzymes produced in Grampositive bacilli and Gram- negative bacteria, lipase and Gram-positive cocci, PULLULANASE, carboxy methyl cellulase. Among these isolates, several strains with the ability to produce different valuable enzymes were selected, including AMB1(8 enzymes); AMB7, AMBI0, AMB12(7 enzymes); AMB5, AMB6, AMB11, AMB13(6 enzymes); AMB2, AMB3, AMB8, AMB9(5 enzymes); AMB4, AMGl(4 enzymes); AMCl, AMC2(3 enzymes); AMC3, AMC8, AMCI7, AMG2 (2 enzymes); AMG3(1 enzymes). Selected strains, after more accurate physiological and biochemical studies were identified regarding phylogeny and molecular characteristics using 16S rRNA technique. Sequencing 16SrRNA was performed to complete the identification process, which Gram-positive bacteria were belong to Halobacillus, Thalassobacillus, Bacillus, Salinicoccus and Gram-negative bacteria were belong to ldiomarina, Salicola and Halomonas.

Production of ten hydrolytic enzymes was qualitatively studied on the haloarchaeal strains isolated from Aran-Bidgol hypersaline lake in the central desert area of Iran. A total of 293 haloarchea strains were selected among 300 extremely halophilic isolated prokaryotes. Accordingly, 142, 141, 128, 64, 38, 16, 7, 3 and 1 archaeal isolates were able to produce DNase, amylase, lipase, inulinase, PULLULANASE, protease, cellulase, chitinase and xylanase, respectively. None was able to produce pectinase activity. Combined hydrolytic activity was also detected in many strains. A total of 0.3 % of the strains showed 6 hydrolytic activities, 0.3 % of the strains had 5 hydrolytic activities, 5.4 % of the strains presented 4 hydrolytic activities, 25 % of the strains presented 3 hydrolytic activities, 28 % of the strains presented 2 hydrolytic activities and 18 % of the strains presented 1 hydrolytic activity. According to their phenotypic characteristics and comparative partial 16 S rRNA sequence analysis, the halophilic strains were all identified as members of family Halobacteriaceae within 12 different taxa from the following genera: Halorubrum, Haloarcula, Natrinema, Halovivax and Natronomonas. Most enzymes production rate was observed in the genera Halorubrum, Haloarcula and Natrinema whereas; there was not any detectable amount of enzyme production in the genera Halovivax and Natronomonas. The most hydrolytic isolate with 6 combinatorial enzyme production belonged to the genus Natrinema. This investigation showed that the extreme halophilic archaea from Aran-Bidgol lake are a potential source of hydrolytic enzyme under stress conditions and may have possess commercial value.

Starch debranching enzymes that merely hydrolyze α-(1→ 6) glycosidic linkages are classified into isoamylases (EC 3. 2. 1. 68) and PULLULANASEs (EC 3. 2. 1. 41). An exception to this definition is amyloPULLULANASE, a type of PULLULANASE that is capable of cleaving both α-(1→ 4) and α-(1→ 6) linkages. AmyloPULLULANASEs are in demand in liquid sugar industries to generate glucose and some other starch derivatives. PULLULANASEs can be used in conjunction with amylases to improve sugar availability during sugar syrup production. Here, a thermophilic Cohnella sp. A01 amyloPULLULANASE (EC 3. 2. 1. 41) gene, namely Coh4159, was PCR amplified and cloned in pET-26b(+) and transformed into BL21(DE3). Recombinant Coh4159 was heterologously expressed in the presence of 0. 5 mM IPTG and purified via affinity chromatography, and further characterized. Enzyme activity was demonstrated via zymogram analysis in the presence of pullulan. The enzyme had a hydrolytic effect on pullulan with Vmax = 2. 85 μ mol. min-1 and Km = 0. 5 mM. Temperature optima and pH were 60 ˚ C and 6. 0, in which the enzyme kept its activity at wide pH (4-9) and temperature (30-70 ˚ C) ranges. The recombinant enzyme kept 50% of its activity for 60 min, 100 min and 120 min when incubated at 80, 70 and 60 ˚ C, respectively. Amongst metal ions tested, Mn2+ and Ca2+ have improved the enzyme activity both at 5 and 10 mM. The results promise the capability of producing a commercial industrial enzyme well-suited to liquid sugar syrup industry specifications.